Influenza is the generic term for diseases or infections caused by the influenza virus. Influenza viruses are members of the Orthomyxoviridae family of viruses and comprise two genera: influenza A and B viruses, and influenza C virus. Influenza A, B and C viruses are distinguished on the basis of their internal nucleoprotein and matrix proteins which are specific for each viral type. Influenza A viruses are naturally able to infect a range of animal species, including humans, swine, birds, seals and horses. Influenza B viruses, however, infect only humans, whilst influenza C virus infects humans and swine. Influenza A viruses are further categorised into subtypes that are determined by the antigenicity of the surface glycoproteins, the haemagglutinin (H) and neuraminidase (N).
Historically, influenza A human infections have been caused by three subtypes of haemagglutinin (H1, H2 and H3) and two neuraminidase subtypes (N1 and N2); more recently human infections by the previously avian-restricted subtypes H5, H7 and H9 have also been reported. A total of 16 distinct haemagglutinin and 9 neuraminidase influenza A subtypes have been identified to date; these are all prevalent in birds. Swine and horses, like humans, are limited to a much narrower range of subtypes.
Influenza A and B virions are pleiomorphic in structure, spherical examples being 80-120 nm in diameter, whilst filamentous forms may be up to 300 nm in length. There are approximately 500 surface spike glycoproteins per particle (usually in the ratio of four to five haemagglutinin proteins to one neuraminidase) that are embedded in a host-derived lipid bilayer membrane. Within the membrane is the transmembrane ion channel protein M2, whilst the structural protein M1 underlies the bilayer. Within the core of the virus, the single stranded negative sense RNA is associated with the six other viral proteins expressed from its genome: the nucleoprotein (NP), three transcriptases (PB2, PB1, and PA) and two nonstructural proteins (NS1 and NS2). The influenza virus genome comprises eight segments; a feature that enables “gene swapping” reassortment. The haemagglutinin enables the virus to bind to host cell receptors and facilitates the entry of the virus into the cell where it will replicate. The neuraminidase protein enzymatically cleaves terminal sialic acid residues, and is believed to assist in the transport of the virus through the mucin layer of the respiratory tract as well as facilitating the budding of the progeny virus away from the host cell. Influenza C viruses, which present much less of a health-risk to humans possess a single surface protein which combines the haemagglutinin, fusion activity and receptor destroying activity.
As a result of the error prone RNA polymerase enzyme, both the haemagglutinin and neuraminidase proteins of the influenza virus are liable to point mutations which need not necessarily affect the ability of the virus to replicate. Such a mutation (or coincident mutations) at one of the sites recognised by the host antibody response may result in the host antibody, induced by vaccination or a previous infection, being unable to bind effectively to the “new” virus strain thereby allowing an infection to persist. As the human influenza strains are continually evolving via these point mutations, the virus is able to escape from the limited antibody repertoire of the human immune response and cause epidemics. The regular “seasonal” bouts of influenza infections are therefore caused by the circulating strains in the population undergoing antigenic drift.
During seasonal epidemics influenza can spread around the world quickly and inflicts a significant economic burden in terms of hospital and other healthcare costs and lost productivity. The virus is transmitted in droplets in the air from human-to-human and targets epithelial cells in the trachea and bronchi of the upper respiratory tract. Influenza virus may also be picked up from contaminated surfaces and passed to the mouth. Disease spreads very quickly especially in crowded circumstances through coughing and sneezing. The stability of the virus is favored by low relative humidity and low temperatures and, as a consequence, seasonal epidemics in temperate areas tend to appear in winter. Greater morbidity and mortality is observed with influenza A strains, with influenza B usually associated with lower attack rates and a milder disease. Occasionally, however, influenza B can cause epidemics of the same severity as type A viruses. Influenza B is primarily a childhood pathogen and does not usually exhibit the same degree of antigenic variation as type A.
The typical uncomplicated influenza infection is characterised by a rapid onset of illness (headache, cough, chills) followed by fever, sore throat, significant myalgias, malaise and loss of appetite. Further symptoms may include rhinorrhoea, substernal tightness and ocular symptoms. The most prominent sign of infection is the fever that is usually in the 38-40° C. temperature range. Whilst the majority of people will recover from influenza infection within one to two weeks without requiring any medical treatment, for certain members of the population the disease may present a serious risk. Such individuals include the very young, the elderly and people suffering from medical conditions such as lung diseases, diabetes, cancer, kidney or heart problems. In this “at risk” population, the infection may lead to severe complications of underlying diseases, bacterial pneumonia, (caused by respiratory pathogens such as Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus) and death. The clinical features of influenza infection are similar in children, although their fever may be higher and febrile convulsions can occur. In addition, children have a higher incidence of vomiting and abdominal pain as well as otitis media complications, croup and myositis.
The World Health Organization estimates that in annual influenza epidemics 5-15% of the population is affected with upper respiratory tract infections. Hospitalization and deaths mainly occur in high-risk groups (elderly and the chronically ill). Although difficult to assess, these annual epidemics are thought to result in between three and five million cases of severe illness and approximately 250 000 and 500 000 deaths every year around the world. Over 90% of the deaths currently associated with influenza in industrialized countries occur among the elderly over 65 years of age. In the U.S.A., the CDC estimate that more than 200,000 people are hospitalized every year on average following complications arising from seasonal influenza infection, with around 36,000 excess mortalities being recorded.
The host immune response that controls the recovery from influenza infection is conferred through a combination of serum antibodies directed to the surface proteins, mucosal secretory IgA antibodies and cell-mediated immune responses. About one to two weeks after a primary infection, neutralizing haemagglutination inhibiting (HAI) antibodies as well as antibodies to neuraminidase are detectable in the serum, peaking at approximately three to four weeks. After re-infection, the antibody response is more rapid. Influenza antibodies may persist for months or years, although in some high-risk groups antibody levels can begin to decline within a few months after vaccination. Secretory IgA antibodies peak approximately 14 days after infection and can be detected in saliva, nasal secretions, sputum and in tracheal washings. Preceding the occurrence of antibody-producing cells, cytotoxic T lymphocytes with specificity for influenza appear, and serve to limit the infection by reducing the maximal viral load whilst mediating more rapid viral clearance through the induction of antiviral cytokines and lysing infected cells. In addition, mononuclear cells infiltrate infected airways providing antibody dependent cell-mediated cytotoxicity against influenza-infected cells.
To date, vaccine approaches against respiratory virus infections such as influenza essentially rely upon the induction of antibodies that protect against viral infection by neutralizing virions or blocking the virus's entry into cells. These humoral immune responses target external viral surface proteins that are conserved for a given strain. Antibody-mediated protection is therefore effective against homologous viral strains but inadequate against heterologous strains with serologically distinct surface proteins. This distinction is of consequence since the surface proteins of many viruses are capable of rapid mutation; for example an effective humoral response-based vaccine against a form of the influenza virus may be ineffective against next season's variant.
There are currently two main types of licensed influenza vaccines. One group of vaccines contains the haemagglutinin and neuraminidase surface proteins of the virus as the active immunogens. These include whole inactivated virus vaccines, split virus vaccines consisting of inactivated virus particles disrupted by detergent treatment, subunit vaccines consisting essentially purified surface proteins from which other virus components have been removed and virosomes where the surface proteins are presented on a liposomal surface. The second group comprises the live attenuated, cold-adapted, strains of virus. For all these vaccines a blend of surface antigens from usually three or four virus strains are required; current commercial influenza vaccines contain antigens from two A subtypes, H3N2 and H1N1, and one type B virus. Each year in September and February respectively, the WHO Global Influenza Program recommends the composition of the influenza vaccine for the next season that normally begins in May-June in the southern hemisphere and in November-December in the northern hemisphere. The composition is based on surveillance data from the worldwide network of national influenza centers and WHO collaborating centers and attempts to cover the likely strains to be circulating nine months later. For this reason, manufacturers are obliged to change the composition of the influenza vaccine on an annual basis in order to ensure an accurate match is achieved with the circulating viral strains.
Most inactivated influenza vaccines are given via the intramuscular route in the deltoid muscle, except in infants where the recommended site is the antero-lateral aspect of the thigh. A single dose of inactivated vaccine annually is appropriate, except for previously unvaccinated preschool children with pre-existing medical conditions who should receive two doses at least one month apart. The live attenuated influenza vaccine (LAIV) is delivered intra-nasally. These have been available in Russia for a number of years and recently licensed for use in the USA in pediatric populations. Such vaccines are able to elicit local antibody and cell-mediated immune responses at the nasal epithelial surface. The live attenuated influenza vaccine is not, however, licensed for use in the USA in elderly populations (over 50 years old).
To enhance the breadth and intensity of the immune response mounted to the influenza virus surface proteins, various adjuvants and alternative immuno-potentiating agents have been evaluated for inclusion in the vaccine formulation. An adjuvant in this context is an agent that is able to modulate the immune response directed to a co-administered antigen while having few if any direct effects when given on its own. Recent licensed developments in the influenza vaccine field include MF-59, a submicron oil-in water emulsion. Aluminium-containing adjuvants are also used by some manufacturers. The intention of these adjuvants is to amplify the resulting serum antibody response to the administered antigens.
Provided there is a good antigenic match between the vaccine strains and those circulating in the general population, inactivated influenza vaccines prevent laboratory-confirmed illness in approximately 70%-90% of healthy adults. However, the CDC highlights that vaccine efficacy in the elderly (over 65 years old) can be as low as 30-40%. Of relevance in this regard is the observation that ageing in humans creates defects in memory T-cell responses that reduce vaccine efficacy and increases the risk to natural infection. Furthermore, a clinical study in a community based setting demonstrated that cell mediated immunity, and not humoral immunity, was correlated with influenza disease protection in a group of over 60 year olds.
In addition, efficacy rates decline significantly if the vaccine strain is antigenically different to the circulating strains. Antigenic variation studies have indicated that four or more amino acid substitutions over at least two antigenic sites of the influenza A haemagglutinin results in a drift variant sufficiently discrete to undermine a vaccine's efficacy (Jin et al. “Two residues in the hemagglutinin of A/Fujian/411/02-like influenza viruses are responsible for antigenic drift from A/Panama/2007/99.” Virology. 2005; 336:113-9). In a case controlled study of adults aged 50-64 years with laboratory confirmed influenza during the 2003-04 season when the vaccine and circulating A/H3N2 strains were not well matched, vaccine effectiveness was estimated to be 52% among healthy individuals and 38% among those with one or more high-risk conditions, according to the CDC. The likelihood of mismatching is raised by the limited manufacturing window of opportunity; the time from strain confirmation, through seed production, antigen manufacture and purification, and the trivalent blending and product filling must all occur in typically less than six months.
Occasionally, a new influenza strain emerges in the population with high pathogenicity and antigenic novelty which results in a worldwide pandemic. Pandemic influenza is the result of an antigenic shift in the surface proteins and represents a serious threat to global health as no pre-existing immunity has been developed by individuals. Pandemic strains are characterised by their sudden emergence in the population and their antigenic novelty. During the twentieth century, four pandemics occurred; in 1918 the causative strain was H1N1, in 1957 H2N2, in 1968 H3N2 and in 1977 H1N1.
There are three alternative explanations for the occurrence of antigenic shift. Firstly, as the influenza virus genome is segmented, it is possible for two influenza strains to exchange their genes upon co-infection of a single host, for example swine, leading to the construction of a replication-competent progeny carrying genetic information of different parental viruses. This process, known as genetic reassortment, is believed to have been the cause of the 1957 and 1968 pandemics. The 1968 pandemic arose when the H3 haemagglutinin gene and one other internal gene from an avian donor reassorted with the N2 neuraminidase and five other genes from the H2N2 human strain that had been in circulation. Secondly, a non-human influenza strain acquires the ability to infect humans. The 1918 pandemic arose when an avian H1N1 strain mutated to enable its rapid and efficient transfer from human-to-human. Thirdly, a strain that had previously caused an epidemic may remain sequestered and unaltered within the human population. The 1977 H1N1 pandemic strain, for example, was essentially identical to a strain that had caused an epidemic 27 years previously and was undetected in the human and animal reservoir over the intervening years.
An influenza pandemic is threatened once three principal criteria have been met:                1. An influenza virus HA subtype, unseen in the human population for at least one generation, emerges (or re-emerges).        2. The virus infects and replicates efficiently in humans, causing significant illness.        3. The virus is transmitted readily and sustainably between humans.        
Global pandemics can afflict between 20% and 40% of the world's population in a single year. The pandemic of 1918-19, for example, affected 200 million people, killing over 30 million worldwide. In the United States, more than half a million individuals died, which represented 0.5% of the population. Although healthcare has dramatically improved since that time, with vaccines and antiviral therapies being developed, the CDC estimate that a pandemic today would result in two to seven million deaths globally.
Since 1999, three different influenza subtype strains (H5N1, H7N7 and H9N2) have crossed from avian species to humans, all causing human mortality. As of Aug. 14, 2007 a total of 320 human cases of H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) infection had been recorded worldwide, with 193 deaths.
Unlike normal seasonal influenza, where infection causes only mild respiratory symptoms in most healthy people, the disease caused by H5N1 follows an unusually aggressive clinical course, with rapid deterioration and high fatality. Primary viral pneumonia and multi-organ failure are common. It is significant that most cases have occurred in previously healthy children and young adults. H5N1 HPAIV incubates longer than other human influenza viruses before causing symptoms, up to eight days in some cases. In household clusters of cases, the time between cases has generally ranged from two to five days but has been reported to take as long as 17 days.
Initial symptoms of H5N1 HPAIV infection are more likely to include diarrhea and can appear up to a week before any respiratory symptoms. This feature, combined with the detection of viral RNA in stool samples, suggests that the virus can multiply in the gastrointestinal tract. Lower respiratory tract symptoms such as shortness of breath appear early in the course of the illness, whereas upper respiratory symptoms such as rhinorrheoa are less common.
H5N1 HPAIV presently meets two of the conditions required for a pandemic; the H5 haemagglutinin represents a new antigen for humans. No one will have immunity should an H5N1-like pandemic virus emerge. In addition, the virus has infected more than 300 humans, with an apparent mortality rate of over 60%.
All prerequisites for the start of a pandemic have therefore been met save one: the establishment of efficient and sustained human-to-human transmission of the virus. The risk that the H5N1 virus will acquire this ability will persist as long as opportunities for human infections occur. This is believed to be a realistic probability, either through step-wise mutation or through reassortment with a human-adapted strain.
At the scientific level, one or more changes to the virus phenotype are necessary before the virus strain could achieve ready human-to-human transmission and begin a pandemic. However, a number of recent observations including specific mutations detected in recent human isolates from Turkey, the increasing pathogenicity to mammals of the circulating virus, the expansion of the H5N1 HPAIV host range to include other mammals, such as tigers and cats that were previously considered to be resistant to infection with avian influenza viruses, all indicate that the H5N1 virus is continuing to evolve capabilities that may ultimately facilitate human-to-human transmission.
Other influenza viruses with possibly even greater pandemic potential may yet emerge. These include a number of H9 and H7 virus strains, which in recent years have also been transmitted to humans. H9 viruses are now endemic in poultry in Asia and also have crossed efficiently into pig populations in South Eastern and Eastern China. Of concern is the fact that the H9N2 strains possess typical human-like receptor specificity and have a broad host range.
In early 2003, an H7N7 HPAIV outbreak occurred in poultry in the Netherlands. Bird-to-human transmission of the H7N7 virus occurred in at least 82 cases. Conjunctivitis was the most common disease symptom in people infected with the H7 strain, with only seven cases displaying typical influenza-like illness. The virus did not prove highly pathogenic for humans and only one fatal case was observed. Other viruses with pandemic potential are those of the H2 subtype, because of its past history as a pandemic virus, and H6 because of its high incidence in poultry species in Asia and North America.
This indicates that a threat of a new human influenza pandemic is not uniquely linked to the emergence of HPAI H5N1.
In preparation for an influenza pandemic a number of clinical trials with candidate H5N1 influenza vaccines have been conducted. These have consistently shown that in order to generate a serum antibody response predicted to be protective, multiple doses of either a much higher amount of haemagglutinin antigen than is normally used in a seasonal vaccine or the inclusion of an adjuvant is required. This is a direct reflection of the immunological naivety of the population to the H5 haemagglutinin. At the present, the only options available for a pandemic influenza vaccine are therefore either one with a very high HA content, which would severely limit the number of doses that could be produced, or the use of an adjuvant that is not currently licensed in the majority of countries. It should also be appreciated that a vaccine that matches the pandemic strain will take many months to manufacture from the time that it is first isolated in humans; a stockpiled vaccine produced in advance of the emergence of a pandemic will most probably not be antigenically identical and therefore provide only limited protection, if any at all. Evidence of antigenic drift is already evident in the most recent outbreaks of H5N1.
In summary, there is a clear requirement for both seasonal and pandemic influenza vaccines to be improved:                1. There are obvious limitations in their efficacy, in particular in unprimed individuals. This is of specific concern with regard to the prospects of an influenza pandemic arising from antigenic shift.        2. The dependence on being able to predict accurately the influenza strains likely to be circulating in the following fall/winter seasons. A mismatch between the vaccine strains and those actually causing infections will render a significant proportion of the population vulnerable to influenza.        3. The need to re-vaccinate at risk groups on a yearly basis as the virus undergoes antigenic drift.        4. Capacity constraints, as there are only a limited number of potential biological manufacturing plants worldwide.        5. The protection afforded to the elderly age group is limited by conventional vaccines.        
Improved classes of influenza vaccine therefore are needed, which are preferably synthetic, stable, and effective against all influenza A strains (including potential pandemic strains), with enhanced efficacy in the elderly (at risk) groups.